@article{ded7ce68906b4c16b6e380cdf2a2c582,
title = "Genetically Targeted Ratiometric and Activated pH Indicator Complexes (TRApHIC) for Receptor Trafficking",
abstract = "Fluorescent protein-based pH sensors are useful tools for measuring protein trafficking through pH changes associated with endo- and exocytosis. However, commonly used pH-sensing probes are ubiquitously expressed with their protein of interest throughout the cell, hindering our ability to focus on specific trafficking pools of proteins. We developed a family of excitation ratiometric, activatable pH responsive tandem dyes, consisting of a pH sensitive Cy3 donor linked to a fluorogenic malachite green acceptor. These cell-excluded dyes are targeted and activated upon binding to a genetically expressed fluorogen-activating protein and are suitable for selective labeling of surface proteins for analysis of endocytosis and recycling in live cells using both confocal and superresolution microscopy. Quantitative profiling of the endocytosis and recycling of tagged β2-adrenergic receptor (B2AR) at a single-vesicle level revealed differences among B2AR agonists, consistent with more detailed pharmacological profiling.",
author = "Perkins, {Lydia A.} and Qi Yan and Schmidt, {Brigitte F.} and Dmytro Kolodieznyi and Saumya Saurabh and Larsen, {Mads Breum} and Watkins, {Simon C.} and Laura Kremer and Bruchez, {Marcel P.}",
note = "Funding Information: *E-mail: bruchez@cmu.edu. ORCID Marcel P. Bruchez: 0000-0002-7370-4848 Author Contributions L.A.P. performed imaging experiments, data analysis, and wrote the paper. Q.Y. performed imaging, photobleaching experiments, initial data analysis, and wrote the paper. B.F.S. synthesized TO1-Cypher5, Cy3−MG, Cy3pH(S/S)−MG, Cy3pH(S/SA)−MG, and Cy3pH(SA/SA)−MG. D.K. developed a quick pipeline for organizing large single-vesicle time course data sets for Prism, collected spectroscopy data, and helped with methods. M.B.L. performed pH calibration and data analysis. S.C.W. performed data analysis and provided assistance with STED microscopy. L.K. and S.S. recorded emission and excitation spectra of Cy3pH(S/S)−MG, Cy3pH-(S/SA)−MG, and Cy3pH(SA/SA)−MG. M.P.B. designed sensors and experiments and wrote the paper. Funding NMR instrumentation at Carnegie Mellon University was partially supported by the National Science Foundation (CHE-0130903 and CHE-1039870). The STED microscope was acquired under National Institutes of Health (NIH) Shared Instrument Program Grant S10OD021540. This work was supported in part by NIH Grants R01EB017268 and U54GM103529. Notes The authors declare the following competing financial interest(s): M.P.B. is founder and Chief Scientific Officer at Sharp Edge Laboratories, Inc., a licensee commercially utilizing the FAP fluorogen technology. Publisher Copyright: {\textcopyright} 2017 American Chemical Society.",
year = "2018",
month = feb,
day = "6",
doi = "10.1021/acs.biochem.7b01135",
language = "English (US)",
volume = "57",
pages = "861--871",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "5",
}